Abstract t Adipose tissue-derived mesenchymal stem cells (AD-MSCs) have the potential to serve as a critical tool for bone tissue engineering as their relatively high frequency in the body can provide the exceedingly large numbers of cells required for regenerative therapies. Unfortunately, many studies have shown that AD-MSCs have considerably less osteogenic potential than other types of stem cells. Our group has developed an in vitro-produced extracellular matrix (ECM) that recapitulates specific attributes of the native bone marrow (BM-) and adipose (AD-) microenvironment. In previous studies, we demonstrated that BM-ECM significantly promotes osteogenic differentiation by bone marrow (BM)-derived MSCs relative to standard culture plastic. In the current proposal, we evaluate the potential of BM-ECM to increase the osteogenic capacity of AD-MSCs, and by doing so, dramatically improve their clinical relevance for skeletal repair and regeneration. Our preliminary data show that BM- and AD-derived ECMs are capable of guiding BM-MSC differentiation towards osteoblast and adipocyte lineages. These ECMs are characterized by significant differences in biochemical composition, architecture and even mechanical properties. We demonstrated that BM- and AD- MSCs cultured on both types of tissue-specific ECM proliferated more readily when each type of MSC was maintained on ECM-derived from the same respective tissue. Additionally, we observed that BM- and AD- MSCs both exhibited low-circularity spreading on BM-ECM, while on AD-ECM, they displayed a high-circularity spreading morphology. These observations are consistent with recent reports demonstrating that changes in MSC morphology are ultimately related to signaling cues involved in lineage-decisions. Furthermore, recent studies have suggested that focal adhesion complex formation can regulate stem cell fate decisions through the canonical Wnt/?-catenin pathway. We will investigate if unique focal adhesion complexes form on tissue- specific ECMs and reconstitute the microenvironment(s) responsible for directing MSC differentiation to either the osteoblast and adipocyte lineage. As a result of these observations, we hypothesize that the bone-specific microenvironment of BM-ECM can re-train AD-MSCs in order to enhance their osteogenic potential. The following specific aims are proposed to test this hypothesis: 1) To determine differences in BM- and AD-MSC differentiation, assessed with assays of osteogenesis and adipogenesis in vitro and in vivo, with culture on tissue culture plastic (TCP) and tissue-specific ECM and 2) To determine the molecular mechanism(s) responsible for promoting MSC differentiation to the osteoblast and adipocyte lineages with culture on TCP and tissue-specific ECM.